Tumor suppressor p53 protects cells from oncogenesis and promotes sensitivity to ant-cancer therapy. p53 protein expression remains low under basal conditions due to MDM2-mediated feedback inhibition and the E3 ubiquitin ligase activity of MDM2 (Wade M et al., Nat Rev Cancer 2013; 13: 83-96). Cellular stresses such as DNA damage or other cellular stress, hypoxia, viral infection or oncogenic transformation stimulate the p53 pathway by stabilizing p53 protein and by inducing its transcriptional activity (Vousden K H, et al. Cell 2009; 137: 413-31).
Active p53 up-regulates the expression of multiple genes, including p21 (el-Deiry W S, et al., Cell 1993; 75: 817-25), Puma (Hikisz P, et al., Cell Mol Biol Lett 2012; 17: 646-69) and DR5 (Wu G S, et al., Adv Exp Med Biol 2000; 465: 143-51), inducing cell cycle arrest, cell death and DNA repair processes to prevent cellular transformation and tumor development. Inactivation of p53 occurs most human tumors due to p53 mutation or down-regulation of p53 expression by various inhibitors, such as MDM2 or HPV E6 (Brooks C L, et al., Mol Cell 2006; 21: 307-15; Olivier M, et al., Cold Spring Harb Perspect Biol 2010; 2: a001008; and Havre P A, et al., Cancer Res 1995; 55: 4420-4). Deregulation of p53 represents a basic difference between normal and cancer cells.
Over two decades ago, wild-type p53 was shown to suppress colon cancer cell colony growth, despite their other genetic alterations including KRAS, PIK3CA and APC mutation as well as metastasis promoting genes (Baker S J, et al., Science 1990; 249: 912-5; and van Oijen M G, et al., Clin Cancer Res 2000; 6: 2138-45). Therefore, p53 pathway restoration remains an outstanding strategy for cancer therapy, and small molecules continue to represent a reasonable and potentially feasible strategy to achieve this goal.
Over 50% of human cancers harbor mutant p53, which inactivates p53 pathway signaling and its tumor suppressor function (van Oijen M G, et al., Clin Cancer Res 2000; 6: 2138-45). The vast majority of p53 mutations in human cancer are missense point mutations. Most mutations are clustered in the p53 DNA-binding domain, which causes mutant p53 to lose its DNA binding and transactivation capability leading to a failure to up-regulate p53 effector genes (Olivier M, et al., Cold Spring Harb Perspect Biol 2010; 2: a001008). p53 DNA mutations not only abrogate the p53 tumor suppressor function, but can also endow mutant p53 with a gain-of-function (GOF), rendering it a proto-oncogene (Muller P A, et al., Nat Cell Biol 2013; 15: 2-8; and Oren M, et al., Cold Spring Harb Perspect Biol 2010; 2: a001107). Delivery of mutant p53 into p53-null tumor cells was found to accelerate cancer development (Dittmer D, et al., Nat Genet 1993; 4: 42-6; and Olive K P, et al., Cell 2004; 119: 847-60), suggesting that GOF can exert oncogenic activity. One property of mutant p53 GOF is to form aberrant protein complexes with numerous interacting protein factors, including a subset of transcription factors such as SP1, NF-Y, p53, and p63/p73, to perturb their activities (Freed-Pastor W A, et al., Genes Dev 2012; 26: 1268-86). For example, inactive p73 may contribute to chemoresistance.
Many studies from animals and cell-based assays suggest that the GOF of mutant p53 contributes to tumorigenesis, tumor progression and resistance to therapy (Oren M, et al., Cold Spring Harb Perspect Biol 2010; 2: a001107). Knocking down mutant p53 has been found to sensitize cancer cells to chemotherapy (Li D, et al., Cell Death Differ 2011; 18: 1904-13; and Wang J, et al., J Cell Biochem 2011; 112: 509-19). Therefore, targeting mutant p53 is an attractive strategy to overcome drug resistance and to sensitize tumors to cancer therapy. This concept is particularly further developed and mechanistically explored in the present study with NSC59984.
Some small molecule compounds targeting mutant p53 have been selected based on putative conformational changes within mutant p53 to restore wild-type p53. For example, CP31398 (Foster B A, et al., Science 1999; 286: 2507-10), PRIMA-1 (Bykov V J, et al., Nat Med 2002; 8: 282-8) and NSC319726 (Yu X, et al., Cancer Cell 2012; 21: 614-25) have been proposed to cause a conformational shift from mutant to wild-type p53, reactivating p53 function in tumor suppression. Although several small molecules can restore the p53 pathway, the GOF of mutant p53 can remain in the tumor cell and can represent an obstacle to tumor suppression as well as therapeutic efficacy. Eliminating mutant p53 is an approach that we decided to pursue in an attempt to abolish the GOF properties of mutant p53 in tumor cells, and with the idea that mutant p53 may represent a challenge for the general approach to stimulate p73, given the ability of mutant p53 to quench the tumor suppressive activity of p73.
A major difference between wild-type and mutant p53 is that mutant p53 proteins are hyper-stabilized in cells. The molecular chaperones Hsp90 and Hsp70 play an important role in keeping mutant p53 protein stable by inhibiting MDM2 and CHIP (Li D, et al., Mol Cancer Res 2011; 9: 577-88). Few compounds have been reported to destabilize mutant p53, including 17AAG, Saha, gambogic acid and Arsenic (Li D, et al., Cell Death Differ 2011; 18: 1904-13; Wang J, et al., J Cell Biochem 2011; 112: 509-19; Li D, et al., Mol Cancer Res 2011; 9: 577-88; and Zhang Y, et al., J Biol Chem 2011; 286: 16218-28). HDAC inhibitors and gambogic acid have been reported to destabilize mutant p53 by activating MDM2 or CHIP (Li D, et al., Cell Death Differ 2011; 18: 1904-13; and Wang J, et al., J Cell Biochem 2011; 112: 509-19). However, those compounds are incapable of restoring the p53 pathway of mutant p53 in tumor cells, and they have many other targets and mechanisms, making them non-specific. Thus, small molecules with the dual capability to restore the p53 pathway and deplete mutant p53 GOF proteins represent a novel strategy for cancer therapy and appear desirable to pursue for further therapeutic development. p73, a member of the p53 family, is a transcription factor with high structural and sequence homology with p53. p73 has been found to have similar functions as wild-type p53 (Melino G, et al., Nat Rev Cancer 2002; 2: 605-15). p73 can transactivate the vast majority of p53 transcriptional target genes by binding to their regulatory regions in the same manner as p53, including in heterologous complexes of p53:p73; thereby impacting on cell growth and cell death pathways (Lunghi P, et al., Clin Cancer Res 2009; 15: 6495-502). Knock-out of p73 contributes to tumor development, functionally implicating p73 as a tumor suppressor (Tomasini R, et al., Genes Dev 2008; 22: 2677-91). Unlike p53, p73 is rarely deleted or mutated in human cancer. p73 is activated by complex signaling pathways in mammalian cells under stress. For example, c-Abl phosphorylates p73 and p300 acetylates p73 in response to cellular stress or DNA damage (Conforti F, et al., Cell Death Dis 2012; 3: e285). Activated p73 induces apoptosis and enhances chemosensitivity. A large variety of chemotherapeutic agents, such as camptothecin, etoposide and cisplatin, can up-regulate p73 expression (Irwin M S, et al., Cancer Cell 2003; 3: 403-10). However, in mutant p53-expressing cancer cells, mutant p53 inhibits p73 activation by binding with p73 to form an inhibited complex with respect to the transactivation of target genes (Freed-Pastor W A, et al., Genes Dev 2012; 26: 1268-86). Inactivated p73 promotes chemoresistance. Therefore, p73 provides a legitimate and bonafide attractive target to restore the p53 pathway in cancer therapy. A peptide, 37AA, has been found to cause p73 dependent cancer cell apoptosis (Bell H S, et al., J Clin Invest 2007; 117: 1008-18). A small molecule, RETRA, was shown to release p73 by disturbing interaction of p73 and mutant p53 (Kravchenko J E, et al., Proc Natl Acad Sci USA 2008; 105: 6302-7). These studies support the strategy of bypassing dysfunctional p53 signaling in cancer therapy through stimulation of p73-dependent signaling, while at the same time attempting to eliminate mutant p53, as is reported here.
There is a need in the art for small molecule compounds that both destabilize mutant p53 and restore wild-type p53 pathway via the activation of p73 in cancer cells. The present invention addresses this unmet need.